Optimized protocol for quantification of extracellular nicotinamide adenine dinucleotide: evaluating clinical parameters and pre-analytical factors for translational research.

Nicotinamide adenine dinucleotide (NAD), a coenzyme for more than 500 enzymes, plays a central role in energy production, metabolism, cellular signaling, and DNA repair. Until recently, NAD was primarily considered to be an intracellular molecule (iNAD), however, its extracellular species (eNAD) has recently been discovered and has since been associated with a multitude of pathological conditions. Therefore, accurate quantification of eNAD in bodily fluids such as plasma is paramount to answer important research questions. In order to create a clinically meaningful and reliable quantitation method, we analyzed the relationship of cell lysis, routine clinical laboratory parameters, blood collection techniques, and pre-analytical processing steps with measured plasma eNAD concentrations. Initially, NAD levels were assessed both intracellularly and extracellularly. Intriguingly, the concentration of eNAD in plasma was found to be approximately 500 times lower than iNAD in peripheral blood mononuclear cells (0.253 ± 0.02 μM vs. 131.8 ± 27.4 μM,  = 0.007, respectively). This stark contrast suggests that cellular damage or cell lysis could potentially affect the levels of eNAD in plasma. However, systemic lactate dehydrogenase in patient plasma, a marker of cell damage, did not significantly correlate with eNAD ( = 33;  = -0.397;  = 0.102). Furthermore, eNAD was negatively correlated with increasing c-reactive protein (CRP,  = 33;  = -0.451;  = 0.020), while eNAD was positively correlated with increasing hemoglobin ( = 33;  = 0.482;  = 0.005). Next, variations in blood drawing, sample handling and pre-analytical processes were examined. Sample storage durations at 4°C (0-120 min), temperature (0° to 25°C), cannula sizes for blood collection and tourniquet times (0 - 120 s) had no statistically significant effect on eNAD ( > 0.05). On the other hand, prolonged centrifugation (> 5 min) and a faster braking mode of the centrifuge rotor (< 4 min) resulted in a significant decrease in eNAD levels ( < 0.05). Taken together, CRP and hemoglobin appeared to be mildly correlated with eNAD levels whereas cell damage was not correlated significantly to eNAD levels. The blood drawing trial did not show any influence on eNAD, in contrast, the preanalytical steps need to be standardized for accurate eNAD measurement. This work paves the way towards robust eNAD measurements, for use in future clinical and translational research, and provides an optimized hands-on protocol for reliable eNAD quantification in plasma.